Candida albicans is the most frequent causative agent of candidiasis, now the third-to-fourth most common infection in hospitals in the US and worldwide, and this opportunistic fungus represents an increasing threat to an ever expanding population of immune- and medically-compromised patients. Candidiasis carries unacceptably high mortality rates, about 30-60%, clearly indicating that current antifungal therapy is still ineffective, due to the limited armamentarium of antifungal agents, the toxicity displayed by some of the current therapies, and the emergence of resistance to most classes of antifungals. Moreover, there are no new effective drugs in sight, and the antifungal pipeline is mostly dry. The ability to form biofilms, microbial communities attached to surfaces, is intimately linked to the pathogenesis of C. albicans, and indeed the majority of manifestations of candidiasis are associated with biofilm formation, which further complicates treatment. As such biofilm formation represents a high value target for the development of novel antifungals. Most recently, we have carried out high content screens and identified small molecule compounds that specifically inhibit C. albicans biofilm formation. We have been developing some of these compounds as novel antifungals, having confirmed their potent biofilm-inhibitory activity, lack of toxicity and efficacy in vivo using relevant animal models of candidiasis. The main goal of the current project is to further advance the development of the current leading chemical series of compounds, for which we propose three specific objectives. i) Medicinal Chemistry. We will engage on a focused and systematic medicinal chemistry campaign to advance 3-4 structurally diverse lead chemical series in parallel, establish structure- activity relationships that improve biofilm inhibition properties while also improving physical chemical drug-like properties. ii) In vivo efficacy. We will test a discrete number of compounds within each chemical series in the murine model of hematogenously disseminated candidiasis, as well as in the murine models of catheter- associated candidiasis and oral candidiasis, both of which are associated with biofilm formation. iii) Insights into mechanisms of action, potential for resistance development, and further in vitro characterization. We will perform further characterization of representative compounds of each chemical series showing the most promise in animal models. These will include RNA-Sequencing and screening of mutant collections to gain insights into the molecular target(-s) and mechanism(-s) of action, serial passage assays to evaluate potential development of resistance, combination with current antifungals, as well as in vitro safety pharmacological profiling for determination of off-target activities.